Using Modified Nucleotides to Map the DNA Determinants of the Tus-TerB Complex, the Protein-DNA Interaction Associated with Termination of Replication in Escherichia coli

Abstract

A series of modified nucleotides was used to map the hydrogen-bonding and hydrophobic sites of the TerB DNA required for Tus interaction. Each of four consensus guanine residues in the TerB-binding site was replaced by 7-deazaguanine, 2-aminopurine, or inosine nucleobase analogues, and each thymine by a uracil analogue. The observable equilibrium dissociation constant for the Tus protein-TerB DNA complex was measured at pH 7.5, 25 degrees C, and 150 mM potassium glutamate using a competition binding method. Substitutions made at position 10 with a 7-deazaguanine, 2-aminopurine, or inosine analogue had a large effect on the stability of the complex, approximately +3 kcal/mol in each case. Substitutions made at positions 13 and 17 had a varied response. For uracil substitutions, potential hydrophobic sites were identified at six positions in the TerB DNA. The energetic penalty for the removal of a single methyl group ranged between +1 and +2 kcal/mol. Rate dissociation measurements agree with these results. Overall, major and minor groove determinants are required for binding. An unusual result was that the conserved nucleotide at position 6 did not significantly affect in vitro binding of the complex.